CRISPR-assisted multi-dimensional regulation for fine-tuning gene expression in Bacillus subtilis

• Published in: Nucleic acids research 2019-04, Vol.47 (7), p.e40-e40
• Main Authors: Lu, Zhenghui, Yang, Shihui, Yuan, Xin, Shi, Yunyun, Ouyang, Li, Jiang, Sijing, Yi, Li, Zhang, Guimin
• Format: Article
• Language: English
• Subjects: Bacillus subtilis - genetics; CRISPR-Associated Protein 9 - metabolism; CRISPR-Cas Systems - genetics; Gene Editing; Gene Expression Regulation, Bacterial; Genes, Bacterial - genetics; Methods Online; Molecular Chaperones - metabolism; Mutation; Promoter Regions, Genetic - genetics; Protein Folding; RNA, Guide, CRISPR-Cas Systems - genetics; Transcription, Genetic; Transformation, Genetic
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkz072

Abstract Fine-tuning of gene expression is crucial for protein expression and pathway construction, but it still faces formidable challenges due to the hierarchical gene regulation at multiple levels in a context-dependent manner. In this study, we defined the optimal targeting windows for CRISPRa and CRISPRi of the dCas9-α/ω system, and demonstrated that this system could act as a single master regulator to simultaneously activate and repress the expression of different genes by designing position-specific gRNAs. The application scope of dCas9-ω was further expanded by a newly developed CRISPR-assisted Oligonucleotide Annealing based Promoter Shuffling (OAPS) strategy, which could generate a high proportion of functional promoter mutants and facilitate the construction of effective promoter libraries in microorganisms with low transformation efficiency. Combing OAPS and dCas9-ω, the influences of promoter-based transcription, molecular chaperone-assisted protein folding and protease-mediated degradation on the expression of amylase BLA in Bacillus subtilis were systematically evaluated, and a 260-fold enhancement of BLA production was obtained. The success of the OAPS strategy and dCas9-ω for BLA production in this study thus demonstrated that it could serve as a powerful tool kit to regulate the expression of multiple genes multi-directionally and multi-dimensionally in bacteria.